RESUMEN
Identification of early perturbations induced in cells from non-cancerous breast tissue is critical for understanding possible breast cancer risk from chemical exposure. We have demonstrated previously that exposure to the ubiquitous xenoestrogens, bisphenol A (BPA) and methyl paraben, promotes the hallmarks of cancer in non-malignant human high-risk donor breast epithelial cells (HRBECs) isolated from several donors. Here we show that terephthalic acid (TPA), a major chemical precursor of polyethylene terephthalate (PET) containers used for the storage of food and beverages, increased the ERα: ERß ratio in multiple HRBEC samples, suggesting an estrogenic effect. Although, like BPA and methyl paraben, TPA also promoted resistance to tamoxifen-induced apoptosis, unlike these chemicals instead of inducing an increased S-phase fraction, TPA treatment arrested cell proliferation. DNA-PK, ATM and members of the MRN complex, known to be involved in DNA damage sensor and effector proteins, were elevated indicating induction of DNA strand breaks. Early DNA damage checkpoint response, mediated through p53/p21, led to G1 arrest in TPA-exposed cells. Removal of TPA from the growth medium resulted in the rapid induction of BCL2, increasing the ratio of anti-: pro-apoptotic proteins, together with overexpression of Cyclin A/CDK2 proteins. Consequently, despite elevated p53(pSer15) and H2AX(pSer139), indicating sustained DNA damage, TPA exposed cells resumed robust growth rates seen prior to TPA exposure. The propensity for the perpetuation of DNA aberrations that activate DNA damage pathways in non-malignant breast cells justifies careful consideration of human exposure to TPA, particularly at vulnerable life stages.
Asunto(s)
Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/patología , Mama/patología , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Ácidos Ftálicos/efectos adversos , Tereftalatos Polietilenos/efectos adversos , Antineoplásicos Hormonales/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Mama/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Depuradores de Radicales Libres/efectos adversos , Humanos , Tamoxifeno/farmacologíaRESUMEN
INTRODUCTION: Triple-negative breast cancer (TNBC) is aggressive and lacks targeted therapies. Phosphatidylinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are frequently activated in TNBC patient tumors at the genome, gene expression and protein levels, and mTOR inhibitors have been shown to inhibit growth in TNBC cell lines. We describe a panel of patient-derived xenografts representing multiple TNBC subtypes and use them to test preclinical drug efficacy of two mTOR inhibitors, sirolimus (rapamycin) and temsirolimus (CCI-779). METHODS: We generated a panel of seven patient-derived orthotopic xenografts from six primary TNBC tumors and one metastasis. Patient tumors and corresponding xenografts were compared by histology, immunohistochemistry, array comparative genomic hybridization (aCGH) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) sequencing; TNBC subtypes were determined. Using a previously published logistic regression approach, we generated a rapamycin response signature from Connectivity Map gene expression data and used it to predict rapamycin sensitivity in 1,401 human breast cancers of different intrinsic subtypes, prompting in vivo testing of mTOR inhibitors and doxorubicin in our TNBC xenografts. RESULTS: Patient-derived xenografts recapitulated histology, biomarker expression and global genomic features of patient tumors. Two primary tumors had PIK3CA coding mutations, and five of six primary tumors showed flanking intron single nucleotide polymorphisms (SNPs) with conservation of sequence variations between primary tumors and xenografts, even on subsequent xenograft passages. Gene expression profiling showed that our models represent at least four of six TNBC subtypes. The rapamycin response signature predicted sensitivity for 94% of basal-like breast cancers in a large dataset. Drug testing of mTOR inhibitors in our xenografts showed 77 to 99% growth inhibition, significantly more than doxorubicin; protein phosphorylation studies indicated constitutive activation of the mTOR pathway that decreased with treatment. However, no tumor was completely eradicated. CONCLUSIONS: A panel of patient-derived xenograft models covering a spectrum of TNBC subtypes was generated that histologically and genomically matched original patient tumors. Consistent with in silico predictions, mTOR inhibitor testing in our TNBC xenografts showed significant tumor growth inhibition in all, suggesting that mTOR inhibitors can be effective in TNBC, but will require use with additional therapies, warranting investigation of optimal drug combinations.
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Antineoplásicos/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Western Blotting , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Hibridación Genómica Comparativa , Análisis Mutacional de ADN , Doxorrubicina/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Células MCF-7 , Ratones , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/genética , Fosforilación/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Sirolimus/análogos & derivados , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Transcriptoma/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Widespread distribution of bisphenol-A (BPA) complicates epidemiological studies of possible carcinogenic effects on the breast because there are few unexposed controls. To address this challenge, we previously developed non-cancerous human high-risk donor breast epithelial cell (HRBEC) cultures, wherein BPA exposure could be controlled experimentally. BPA consistently induced activation of the mammalian target of rapamycin (mTOR) pathway--accompanied by dose-dependent evasion of apoptosis and increased proliferation--in HRBECs from multiple donors. Here, we demonstrate key molecular changes underlying BPA-induced cellular reprogramming. In 3/3 BPA-exposed HRBEC cell lines, and in T47D breast cancer cells, proapoptotic negative regulators of the cell cycle (p53, p21(WAF1) and BAX) were markedly reduced, with concomitant increases in proliferation-initiating gene products (proliferating cell nuclear antigen, cyclins, CDKs and phosphorylated pRb). However, simultaneous exposure to BPA and the polyphenol, curcumin, partially or fully reduced the spectrum of effects associated with BPA alone, including mTOR pathway proteins (AKT1, RPS6, pRPS6 and 4EBP1). BPA exposure induced an increase in the ERα (Estrogen Receptor): ERß ratio--an effect also reversed by curcumin (analysis of variance, P < 0.02 for all test proteins). At the functional level, concurrent curcumin exposure reduced BPA-induced apoptosis evasion and rapid growth kinetics in all cell lines to varying degrees. Moreover, BPA extended the proliferation potential of 6/6 primary finite-life HRBEC cultures--another effect reduced by curcumin. Even after removal of BPA, 1/6 samples maintained continuous growth--a hallmark of cancer. We show that BPA exposure induces aberrant expression of multiple checkpoints that regulate cell survival, proliferation and apoptosis and that such changes can be effectively ameliorated.
Asunto(s)
Apoptosis , Proliferación Celular/efectos de los fármacos , Células Epiteliales/fisiología , Glándulas Mamarias Humanas/patología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Compuestos de Bencidrilo/farmacología , Neoplasias de la Mama , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Curcumina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/metabolismo , Estrógenos no Esteroides/farmacología , Femenino , Humanos , Hidroxitestosteronas/farmacología , Cinética , Fenoles/farmacología , Cultivo Primario de Células , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Estrogenic overstimulation is carcinogenic to the human breast. Personal care products (PCPs) commonly contain xenoestrogens (XE), such as parabens and phthalates. Here, we identified the adverse effects of persistent exposure to such PCPs directly within human estrogen responsive breast tissue of subjects enrolled in a regimen of reduced XE use (REDUXE). Pre- and post-intervention fine needle aspirates (FNAs) of the breast were collected from healthy volunteers who discontinued the use of paraben and phthalate containing PCPs over a 28 d period. Based on high-dimensional gene expression data of matched FNA pairs of study subjects, we demonstrate a striking reversal of cancer-associated phenotypes, including the PI3K-AKT/mTOR pathway, autophagy, and apoptotic signaling networks within breast cells of REDUXE compliant subjects. These, and other altered phenotypes were detected together with a significant reduction in urinary parabens and phthalate metabolites. Moreover, in vitro treatment of paired FNAs with 17ß-estradiol (E2), displayed a 'normalizing' impact of REDUXE on gene expression within known E2-modulated pathways, and on functional endpoints, including estrogen receptor alpha: beta ratio, and S-phase fraction of the cell cycle. In a paradigm shifting approach facilitated by community-based participatory research, REDUXE reveals unfavorable consequences from exposure to XEs from daily-use PCPs. Our findings illustrate the potential for REDUXE to suppress pro-carcinogenic phenotypes at the cellular level towards the goal of breast cancer prevention.
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Neoplasias , Ácidos Ftálicos , Humanos , Parabenos , Fosfatidilinositol 3-Quinasas , FenotipoRESUMEN
Breast cancer is an estrogen-driven disease. Consequently, hormone replacement therapy correlates with disease incidence. However, increasing male breast cancer rates over the past three decades implicate additional sources of estrogenic exposure including wide spread estrogen-mimicking chemicals or xenoestrogens (XEs), such as bisphenol-A (BPA). By exposing renewable, human, high-risk donor breast epithelial cells (HRBECs) to BPA at concentrations that are detectable in human blood, placenta and milk, we previously identified gene expression profile changes associated with activation of mammalian target of rapamycin (mTOR) pathway genesets likely to trigger prosurvival changes in human breast cells. We now provide functional validation of mTOR activation using pairwise comparisons of 16 independent HRBEC samples with and without BPA exposure. We demonstrate induction of key genes and proteins in the PI3K-mTOR pathway--AKT1, RPS6 and 4EBP1 and a concurrent reduction in the tumor suppressor, phosphatase and tensin homolog gene protein. Altered regulation of mTOR pathway proteins in BPA-treated HRBECs led to marked resistance to rapamycin, the defining mTOR inhibitor. Moreover, HRBECs pretreated with BPA, or the XE, methylparaben (MP), surmounted antiestrogenic effects of tamoxifen showing dose-dependent apoptosis evasion and induction of cell cycling. Overall, XEs, when tested in benign breast cells from multiple human subjects, consistently initiated specific functional changes of the kind that are attributed to malignant onset in breast tissue. Our observations demonstrate the feasibility of studying renewable human samples as surrogates and reinforce the concern that BPA and MP, at low concentrations detected in humans, can have adverse health consequences.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Fenoles/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Compuestos de Bencidrilo , Western Blotting , Neoplasias de la Mama/genética , Estudios de Casos y Controles , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Estrógenos no Esteroides/farmacología , Femenino , Humanos , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Serina-Treonina Quinasas TOR/genéticaRESUMEN
As a continuous source of hormonal stimulation, environmentally ubiquitous estrogenic chemicals, ie, xenoestrogens (XEs), are a potential risk factor for breast carcinogenesis. Given their wide distribution in the environment and the fact that bisphenol-A (BPA), methylparaben (MP), and perfluorooctanoic acid (PFOA) are uniformly detected in unselected body fluid samples, it must be assumed that humans are simultaneously exposed to these chemicals almost daily. We studied the effects of a ternary mixture of BPA, MP, and PFOA on benign breast epithelial cells at the range of concentrations observed for single chemicals in human samples. Measurements of exposure impact relevant to the breast were based on endpoints associated with "hallmarks" of cancer and "key characteristics" of carcinogens. These included modulation of total estrogen receptor (ER)α, phosphorylated ERα (pERα), total ERß, S-phase induction, and apoptotic evasion. Data from live cell measurements were fit to a log-linear dose-response model. Concentration-dependent reduction of ERß and apoptosis evasion was observed concurrently with the induction of ERα, pERα, and S-phase fraction, and an increased rate of cell proliferation. Beyond additive effects predicted by the sum of individual test XEs, mixture treatment demonstrated synergism for the ERß and apoptosis suppression phenotypes (p > .001). Nonmalignant breast cells were more sensitive than commonly used breast cancer lines to XE treatment in 3 of 5 endpoints. All observations were validated with cells isolated from the normal breast tissue of 14 individuals. At relatively low concentrations, a chemical mixture has striking effects on normal cell function that are missed by evaluation of single components.
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Apoptosis/efectos de los fármacos , Mama/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Xenobióticos/administración & dosificación , Xenobióticos/toxicidad , Compuestos de Bencidrilo/administración & dosificación , Compuestos de Bencidrilo/toxicidad , Mama/metabolismo , Mama/patología , Caprilatos/administración & dosificación , Caprilatos/toxicidad , Línea Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fluorocarburos/administración & dosificación , Fluorocarburos/toxicidad , Humanos , Parabenos/administración & dosificación , Parabenos/toxicidad , Fenoles/administración & dosificación , Fenoles/toxicidadRESUMEN
INTRODUCTION: Age is one of the most important risk factors for human malignancies, including breast cancer; in addition, age at diagnosis has been shown to be an independent indicator of breast cancer prognosis. Except for inherited forms of breast cancer, however, there is little genetic or epigenetic understanding of the biological basis linking aging with sporadic breast cancer incidence and its clinical behavior. METHODS: DNA and RNA samples from matched estrogen receptor (ER)-positive sporadic breast cancers diagnosed in either younger (age
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Envejecimiento/fisiología , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Perfilación de la Expresión Génica , Genoma Humano , Neoplasias Hormono-Dependientes/genética , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Neoplasias Hormono-Dependientes/metabolismo , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Receptor ErbB-2/metabolismo , Receptores de EstrógenosRESUMEN
BACKGROUND: Signaling pathways that converge on two different transcription factor complexes, NFkappaB and AP-1, have been identified in estrogen receptor (ER)-positive breast cancers resistant to the antiestrogen, tamoxifen. METHODS: Two cell line models of tamoxifen-resistant ER-positive breast cancer, MCF7/HER2 and BT474, showing increased AP-1 and NFkappaB DNA-binding and transcriptional activities, were studied to compare tamoxifen effects on NFkappaB and AP-1 regulated reporter genes relative to tamoxifen-sensitive MCF7 cells. The model cell lines were treated with the IKK inhibitor parthenolide (PA) or the proteasome inhibitor bortezomib (PS341), alone and in combination with tamoxifen. Expression microarray data available from 54 UCSF node-negative ER-positive breast cancer cases with known clinical outcome were used to search for potential genes signifying upregulated NFkappaB and AP-1 transcriptional activity in association with tamoxifen resistance. The association of these genes with patient outcome was further evaluated using node-negative ER-positive breast cancer cases identified from three other published data sets (Rotterdam, n = 209; Amsterdam, n = 68; Basel, n = 108), each having different patient age and adjuvant tamoxifen treatment characteristics. RESULTS: Doses of parthenolide and bortezomib capable of sensitizing the two endocrine resistant breast cancer models to tamoxifen were capable of suppressing NFkappaB and AP-1 regulated gene expression in combination with tamoxifen and also increased ER recruitment of the transcriptional co-repressor, NCoR. Transcript profiles from the UCSF breast cancer cases revealed three NFkappaB and AP-1 upregulated genes--cyclin D1, uPA and VEGF--capable of dichotomizing node-negative ER-positive cases into early and late relapsing subsets despite adjuvant tamoxfien therapy and most prognostic for younger age cases. Across the four independent sets of node-negative ER-positive breast cancer cases (UCSF, Rotterdam, Amsterdam, Basel), high expression of all three NFkappaB and AP-1 upregulated genes was associated with earliest metastatic relapse. CONCLUSION: Altogether, these findings implicate increased NFkappaB and AP-1 transcriptional responses with tamoxifen resistant breast cancer and early metastatic relapse, especially in younger patients. These findings also suggest that agents capable of preventing NFkappaB and AP-1 gene activation may prove useful in restoring the endocrine responsiveness of such high-risk ER-positive breast cancers.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/genética , Antagonistas de Estrógenos/farmacología , FN-kappa B/genética , Factor de Transcripción AP-1/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Antagonistas de Estrógenos/uso terapéutico , Humanos , FN-kappa B/metabolismo , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Factor de Transcripción AP-1/metabolismoRESUMEN
Allelic losses characteristic of tumor cells, when displayed by morphologically normal terminal ductal lobular units (TDLUs) adjacent to carcinoma [G. Deng et al., Science (Wash. DC), 274: 2057-2059, 1996], may indicate an extended field of increased cancer susceptibility within the affected breast tissue. We investigated this possibility by asking whether the presence of loss of heterozygosity (LOH) at chromosome 3p11-26 in histologically normal TDLUs (3pLOHn) could lead to an increased risk of local tumor recurrence. We assessed LOHs in normal TDLUs adjacent to 48 informative cases of early-stage invasive breast cancer samples and found 3pLOHn in approximately 25% (13 of 48) of patients whose tumors had 3pLOH in this region. Our analyses suggest that the most frequent region of LOH is localized at 3p24.3. We also demonstrate, using a Cox proportional hazards regression model, that the presence of 3pLOHn was the only variable significantly related to local tumor recurrence, leading to a 3.9-5.2-fold increase in the hazard ratio (P < 0.05). The time to recurrence was longer in such cases than in those without 3pLOHn, suggesting de novo tumor development. These data provide a strong rationale to assess histologically normal breast tissue at the margins of surgically excised cancers for molecular predictors of local recurrence after breast-conserving treatment.
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Neoplasias de la Mama/genética , Pérdida de Heterocigocidad , Recurrencia Local de Neoplasia/genética , Adulto , Anciano , Anciano de 80 o más Años , Mama/fisiología , Mama/ultraestructura , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Cromosomas Humanos Par 3/genética , Femenino , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Proyectos PilotoRESUMEN
Toward the goal of identifying early genetic losses, which mediate the release of human breast epithelium from replicative suppression leading to cellular immortalization, we have used a newly developed in vitro model system. This system consists of epithelial cultures derived from noncancerous breast tissue, treated with the chemical carcinogen N-ethyl-N-nitrosourea, and continuously passaged to yield cell populations culminating in the immortal phenotype. Genome-wide allelotyping of early passage N-ethyl-N-nitrosourea-exposed cell populations revealed aberrations at >10% (18 of 169) loci examined. Allelic losses encompassing chromosomes 6q24-6q27, implicating immortalization-associated candidate genes, hZAC and SEN6, occurred in two independently derived cell lines before the Hayflick limit. Additional LOH sites were present in one cell line at 3p11-3p26, 11p15, and 20p12-13. Allelic losses reported in this cell line preceded detectable levels of telomerase activity and the occurrence of p53-related aberrations. Information gained from the search for early immortalization-associated genetic deletions in cultured cells was applied in a novel approach toward the analysis of morphologically normal terminal ductal lobular units microdissected from 20 cases of ductal carcinoma in situ. Notably, clonal allelic losses at chromosome 3p24 and 6q24 were an early occurrence in adjoining terminal ductal lobular units of a proportion of primary tumors, which displayed loss of heterozygosity (3 of 11 and 3 of 6, respectively). The biological insights provided by the new model system reported here strongly suggest that early allelic losses delineated in immortalized cultures and validated in vivo could serve as surrogate endpoints to assist in the identification and intervention of high-risk benign breast tissue, which sustains the potential for continuous proliferation.
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Alelos , Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Mama/citología , Mama/efectos de los fármacos , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/patología , Adhesión Celular/fisiología , División Celular/fisiología , Línea Celular Transformada , Progresión de la Enfermedad , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Etilnitrosourea , Eliminación de Gen , Genes p16 , Genes p53 , Humanos , Pérdida de Heterocigocidad , Telomerasa/metabolismo , Telómero/fisiologíaRESUMEN
Loss of heterozygosity within the short arm of chromosome 3 is a common molecular event in several types of solid tumors. In breast cancer, 3p loss of heterozygosity occurs in invasive tumor cells as well as in morphologically normal terminal ductal lobular units adjacent to carcinoma in some cases [G. Deng et al., Science (Wash. DC), 274: 2057-2059, 1996.]. The most frequent region of allelic loss at 3p24.3 in morphologically normal terminal ductal lobular units encompasses the thyroid hormone receptor beta1 (TRbeta1) gene. Here we have observed a variable degree of TRbeta1 promoter hypermethylation in all 11 cases of primary breast cancer examined. Moreover, hypermethylation occurred at the same CpG sites in nonmalignant tissue peripheral to carcinoma in 4 of 11 cases. The lack of TRbeta1 nuclear staining, a likely result of biallelic gene inactivation, was observed in 25% (22 of 85) of primary tumors. This is a first demonstration of promoter hypermethylation and a concurrent reduction of TRbeta1 transcripts in breast cancer cell lines, although specific CpG sites targeted for gene silencing remain to be determined. Gene expression was restored by treatment with 5-aza-deoxycytidine in such cases. The observation of early, frequent, and multiple mechanisms of TRbeta1 inactivation suggests a potential role for this gene in the suppression of breast tumorigenesis.
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Alelos , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Silenciador del Gen , Receptores de Hormona Tiroidea/genética , Receptores beta de Hormona Tiroidea , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Metilación de ADN , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Pérdida de Heterocigocidad , Estadificación de Neoplasias , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Hormona Tiroidea/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Invasive lobular and ductal breast tumors have distinct histologies and clinical presentation. Other than altered expression of E-cadherin, little is known about the underlying biology that distinguishes the tumor subtypes. We used cDNA microarrays to identify genes differentially expressed between lobular and ductal tumors. Unsupervised clustering of tumors failed to distinguish between the two subtypes. Prediction analysis for microarrays (PAM) was able to predict tumor type with an accuracy of 93.7%. Genes that were significantly differentially expressed between the two groups were identified by MaxT permutation analysis using t tests (20 cDNA clones and 10 unique genes), significance analysis for microarrays (33 cDNA clones and 15 genes, at an estimated false discovery rate of 2%), and PAM (31 cDNAs and 15 genes). There were 8 genes identified by all three of these related methods (E-cadherin, survivin, cathepsin B, TPI1, SPRY1, SCYA14, TFAP2B, and thrombospondin 4), and an additional 3 that were identified by significance analysis for microarrays and PAM (osteopontin, HLA-G, and CHC1). To validate the differential expression of these genes, 7 of them were tested by real-time quantitative PCR, which verified that they were differentially expressed in lobular versus ductal tumors. In conclusion, specific changes in gene expression distinguish lobular from ductal breast carcinomas. These genes may be important in understanding the basis of phenotypic differences among breast cancers.
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Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patología , Diagnóstico Diferencial , Femenino , Perfilación de la Expresión Génica , Humanos , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Current approaches to chemical screening, prioritization, and assessment are being reenvisioned, driven by innovations in chemical safety testing, new chemical regulations, and demand for information on human and environmental impacts of chemicals. To conceptualize these changes through the lens of a prevalent disease, the Breast Cancer and Chemicals Policy project convened an interdisciplinary expert panel to investigate methods for identifying chemicals that may increase breast cancer risk. METHODS: Based on a review of current evidence, the panel identified key biological processes whose perturbation may alter breast cancer risk. We identified corresponding assays to develop the Hazard Identification Approach for Breast Carcinogens (HIA-BC), a method for detecting chemicals that may raise breast cancer risk. Finally, we conducted a literature-based pilot test of the HIA-BC. RESULTS: The HIA-BC identifies assays capable of detecting alterations to biological processes relevant to breast cancer, including cellular and molecular events, tissue changes, and factors that alter susceptibility. In the pilot test of the HIA-BC, chemicals associated with breast cancer all demonstrated genotoxic or endocrine activity, but not necessarily both. Significant data gaps persist. CONCLUSIONS: This approach could inform the development of toxicity testing that targets mechanisms relevant to breast cancer, providing a basis for identifying safer chemicals. The study identified important end points not currently evaluated by federal testing programs, including altered mammary gland development, Her2 activation, progesterone receptor activity, prolactin effects, and aspects of estrogen receptor ß activity. This approach could be extended to identify the biological processes and screening methods relevant for other common diseases.
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Neoplasias de la Mama/inducido químicamente , Carcinógenos/toxicidad , Estrógenos/toxicidad , Mutágenos/toxicidad , Bioensayo , Daño del ADN , Femenino , Humanos , Proyectos Piloto , Riesgo , Pruebas de ToxicidadRESUMEN
BACKGROUND: To identify the spectrum of malignant attributes maintained outside the host environment, we have compared global gene expression in primary breast tumors and matched short-term epithelial cultures. RESULTS: In contrast to immortal cell lines, a characteristic 'limited proliferation' phenotype was observed, which included over expressed genes associated with the TGFbeta signal transduction pathway, such as SPARC, LOXL1, RUNX1, and DAPK1. Underlying this profile was the conspicuous absence of hTERT expression and telomerase activity, a significant increase in TbetaRII, its cognate ligand, and the CDK inhibitor, p21CIP1/WAF1. Concurrently, tumor tissue and primary cultures displayed low transcript levels of proliferation-related genes, such as, TOP2A, ANKT, RAD51, UBE2C, CENPA, RRM2, and PLK. CONCLUSIONS: Our data demonstrate that commonly used immortal cell lines do not reflect some aspects of tumor biology as closely as primary tumor cell cultures. The gene expression profile of malignant tissue, which is uniquely retained by cells cultured on solid substrates, could facilitate the development and testing of novel molecular targets for breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Células Tumorales Cultivadas/metabolismo , Mama/citología , Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral/citología , Línea Celular Tumoral/metabolismo , Proliferación Celular , Regulación hacia Abajo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/metabolismo , Células Tumorales Cultivadas/citología , Regulación hacia ArribaRESUMEN
Normal cellular phenotypes that serve an oncogenic function during tumorigenesis are potential candidates for cancer targeting drugs. Within a subset of invasive primary breast carcinoma, we observed relatively abundant expression of Tetherin, a cell surface protein encoded by the Bone Marrow Stromal Cell Antigen (BST2) known to play an inhibitory role in viral release from infected immune cells of the host. Using breast cancer cell lines derived from low and intermediate histopathologic grade invasive primary tumors that maintain growth-suppressive TGFß signaling, we demonstrate that BST2 is negatively regulated by the TGFß axis in epithelial cells. Binding of the transcription factor AP2 to the BST2 promoter was attenuated by inhibition of the TGFß pathway thereby increasing BST2 expression in tumor cells. In contrast, inherent TGFß resistance characteristic of high grade breast tumors is a key factor underlying compromised BST2 regulation, and consequently its constitutive overexpression relative to non-malignant breast epithelium, and to most low and intermediate grade cancer cells. In both 2-dimensional and 3-dimensional growth conditions, BST2-silenced tumor cells displayed an enhancement in tamoxifen or staurosporine-induced apoptotic cell death together with a reduction in the S-phase fraction compared to BST2 overexpressing counterparts. In a subset of breast cancer patients treated with pro apoptotic hormonal therapy, BST2 expression correlated with a trend for poor clinical outcome, further supporting its role in conferring an anti apoptotic phenotype. Similar to the effects of gene manipulation, declining levels of endogenous BST2 induced by the phytoalexin - resveratrol, restored apoptotic function, and curbed cell proliferation. We provide evidence for a direct approach that diminishes aberrant BST2 expression in cancer cells as an early targeting strategy to assist in surmounting resistance to pro apoptotic therapies.
Asunto(s)
Antígenos CD/genética , Apoptosis , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Antígenos CD/metabolismo , Antineoplásicos Hormonales/farmacología , Secuencia de Bases , Sitios de Unión , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/mortalidad , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Humanos , Estimación de Kaplan-Meier , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Modelos de Riesgos Proporcionales , Unión Proteica , Resveratrol , Estilbenos/farmacología , Tamoxifeno/farmacología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
BACKGROUND: To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. CONCLUSIONS/SIGNIFICANCE: For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.
Asunto(s)
Neoplasias de la Mama , Regulación Neoplásica de la Expresión Génica , Células Neoplásicas Circulantes , Análisis de la Célula Individual/instrumentación , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Linfoma/sangre , Análisis por Micromatrices/métodos , Técnicas Analíticas Microfluídicas , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
Whether stromal components facilitate growth, invasion, and dissemination of cancer cells or suppress neoplastic lesions from further malignant progression is a continuing conundrum in tumor biology. Conceptualizing a dynamic picture of tumorigenesis is complicated by inter-individual heterogeneity. In the post genomic era, unraveling such complexity remains a challenge for the cancer biologist. Towards establishing a functional association between cellular crosstalk and differential cancer aggressiveness, we identified a signature of malignant breast epithelial response to stromal signaling. Proximity to fibroblasts resulted in gene transcript alterations of >2-fold for 107 probes, collectively designated as Fibroblast Triggered Gene Expression in Tumor (FTExT). The hazard ratio predicted by the FTExT classifier for distant relapse in patients with intermediate and high grade breast tumors was significant compared to routine clinical variables (dataset 1, nâ=â258, HR--2.11, 95% CI 1.17-3.80, p-value 0.01; dataset 2, nâ=â171, HR--3.07, 95% CI 1.21-7.83, p-value 0.01). Biofunctions represented by FTExT included inflammatory signaling, free radical scavenging, cell death, and cell proliferation. Unlike genes of the 'proliferation cluster', which are overexpressed in aggressive primary tumors, FTExT genes were uniquely repressed in such cases. As proof of concept for our correlative findings, which link stromal-epithelial crosstalk and tumor behavior, we show a distinctive differential in stromal impact on prognosis-defining functional endpoints of cell cycle progression, and resistance to therapy-induced growth arrest and apoptosis in low vs. high grade cancer cells. Our experimental data thus reveal aspects of 'paracrine cooperativity' that are exclusively contingent upon the histopathologically defined grade of interacting tumor epithelium, and demonstrate that epithelial responsiveness to the tumor microenvironment is a deterministic factor underlying clinical outcome. In this light, early attenuation of epithelial-stromal crosstalk could improve the management of cases prone to be clinically challenging.
Asunto(s)
Neoplasias de la Mama/patología , Transducción de Señal , Células del Estroma/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patologíaRESUMEN
Inherent cancer phenotypes that are independent of fluctuating cross-talk with the surrounding tissue matrix are highly desirable candidates for targeting tumor cells. Our novel study design uses epithelial cell lines derived from low versus high histologic grade primary breast cancer to effectively diminish the breadth of transient variability generated within the tumor microenvironment of the host, revealing a "paracrine-independent expression of grade-associated" (PEGA) gene signature. PEGA members extended beyond "proliferation-driven" signatures commonly associated with aggressive, high-grade breast cancer. The calcium-binding protein S100P was prominent among PEGA genes overexpressed in high-grade tumors. A three-member fingerprint of S100P-correlated genes, consisting of GPRC5A, FXYD3, and PYCARD, conferred poor outcome in multiple breast cancer data sets, irrespective of estrogen receptor status but dependent on tumor size (P < 0.01). S100P silencing markedly diminished coregulated gene transcripts and reversed aggressive tumor behavior. Exposure to pathway-implicated agents, including the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, phenothiazine, and chlorpromazine, resulted in rapid apoptotic cell death in high-grade tumor cells resistant to the chemotherapeutic drug cisplatin. This is the first comprehensive study describing molecular phenotypes intimately associated with histologic grade whose expression remains relatively fixed despite an unavoidably changing environment to which tumor cells are invariably exposed.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adulto , Anciano , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Tumorales CultivadasRESUMEN
Breast cancer outcome is highly variable. Whether inadvertent exposure to environmental xenobiotics evokes a biological response promoting cancer aggressiveness and a higher probability of tumor recurrence remains unknown. To determine specific molecular alterations which arise in high-risk breast tissue in the presence of the ubiquitous xenoestrogen, bisphenol A (BPA), we used nonmalignant random periareolar fine-needle aspirates in a novel functional assay. Early events induced by BPA in epithelial-stromal cocultures derived from the contralateral tissue of patients with breast cancer included gene expression patterns which facilitate apoptosis evasion, endurance of microenvironmental stress, and cell cycle deregulation without a detectable increase in cell numbers. This BPA response profile was significantly associated with breast tumors characterized by high histologic grade (P < 0.001) and large tumor size (P = 0.002), resulting in decreased recurrence-free patient survival (P < 0.001). Our assays show a biological "fingerprint" of probable prior exposure to endocrine-disrupting agents, and suggest a scenario in which their presence in the microenvironmental milieu of high-risk breast tissue could play a deterministic role in establishing and maintaining tumor aggressiveness and poor patient outcome.
Asunto(s)
Neoplasias de la Mama/patología , Fenoles/farmacología , Adulto , Compuestos de Bencidrilo , Biopsia con Aguja Fina , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Células Epiteliales/patología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
In the search for early deletion targets in sporadic breast cancer, the analysis of TP53, BRCA1, BRCA2, and ATM revealed loss of heterozygosity (LOH) in tumor cells at 1 or more genes in 18 of 24 cases examined. Notably, in more than 60% of such tumors, LOH was detectable in morphologically normal terminal ductal lobular units (TDLUs) adjacent to carcinoma (LOHint). At BRCA2 and ATM, LOHint was most frequent, particularly in TDLUs of women Asunto(s)
Neoplasias de la Mama/genética
, Células Epiteliales/metabolismo
, Predisposición Genética a la Enfermedad
, Adulto
, Proteínas de la Ataxia Telangiectasia Mutada
, Proteína BRCA2/genética
, Mama/metabolismo
, Neoplasias de la Mama/metabolismo
, Carcinoma/genética
, Proteínas de Ciclo Celular
, Aberraciones Cromosómicas
, Daño del ADN
, Reparación del ADN
, Proteínas de Unión al ADN
, Femenino
, Genes BRCA1
, Genes erbB-2/genética
, Genes p53/genética
, Humanos
, Pérdida de Heterocigocidad
, Persona de Mediana Edad
, Reacción en Cadena de la Polimerasa
, Lesiones Precancerosas
, Proteínas Serina-Treonina Quinasas/genética
, ARN/metabolismo
, Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
, Factores de Tiempo
, Proteínas Supresoras de Tumor